Design, Synthesis and Fungicidal Activity of New 1,2,4-Triazole Derivatives Containing Oxime Ether and Phenoxyl Pyridinyl Moiety

A series of novel 1,2,4-triazole derivatives containing oxime ether and phenoxy pyridine moiety were designed and synthesized. The new compounds were identified by nuclear magnetic resonance (NMR) spectroscopy and high-resolution mass spectrometry (HRMS). Compound (Z)-1-(6-(4-nitrophenoxy)pyridin-3-yl)-2-(1H-1,2,4-triazol-1-yl)ethan-1-one O-methyl oxime (5a18) was further confirmed by X-ray single crystal diffraction. Their antifungal activities were evaluated against eight phytopathogens. The in vitro bioassays indicated that most of the title compounds displayed moderate to high fungicidal activities. Compound (Z)-1-(6-(4-bromo-2-chlorophenoxy)pyridin-3-yl)-2-(1H-1,2,4-triazol-1-yl)ethan-1-one O-methyl oxime (5a4) exhibited a broad-spectrum antifungal activities with the EC50 values of 1.59, 0.46, 0.27 and 11.39 mg/L against S. sclerotiorum, P. infestans, R. solani and B. cinerea, respectively. Compound (Z)-1-(6-(2-chlorophenoxy)pyridin-3-yl)-2-(1H-1,2,4-triazol-1-yl)ethan-1-one O-benzyl oxime (5b2) provided the lowest EC50 value of 0.12 mg/L against S. sclerotiorum, which were comparable to the commercialized difenoconazole. Moreover, homologous modeling and molecular docking disclosed possible binding modes of compounds 5a4 and 5b2 with CYP51. This work provided useful guidance for the discovery of new 1,2,4-triazole fungicides.


Crystal structure determination for compound 5a18
Compound 5a18 was recrystallized by slow evaporation from a mixed solvent of methanol/ethyl acetate (1:4 v/v) to afford crystals suitable for X-ray diffraction analysis. Colorless pieces of 5a18 were mounted on a quartz fiber. Cell dimensions and intensities were measured using a Bruker D8 Venture X-ray CMOS diffractometer with graphite monochromated MoKa radiation. The structure was resolved by direct methods with SHELXS-97. Hydrogen atoms were observed and refined at a fixed value of their isotropic displacement parameter. Crystallographic data for the structure of compound 5a18 have been deposited in the Cambridge Crystallographic Data Centre (deposition number CCDC-2015868). Table S1. Crystal data and structure refinement for compound 5a18.

Fungicidal Activity assay (Growth Inhibition Test).
The in vitro fungicidal activities against plant pathogens were tested according to the reported method 2 . The medium was amended with aliquots of each tested compound solution to provide a concentration of 50 mg/L. The tested compounds were dissolved in 0.3 mL of dimethyl sulfoxide (DMSO) and added aseptically to molten agar after autoclaving, when the agar had cooled to 45−50 °C. The concentration of solvent never exceeded 0.1 mg/L. The mixed medium without sample was used as the blank control. The inocula, 5 mm in diameter, were removed from the margins of actively growing colonies of mycelium, placed in the centers of the above plates. Three replicates were done for each concentration, and the control plates were sealed with parafilm and incubated at 26 °C in darkness. The diameter of the mycelium was measured after several days. The inhibition percent was used to describe the control efficiency of the compounds. Inhibition percent (%) = (hyphal diameter in the control − hyphal diameter in the treatment)/hyphal diameter in the control. The results are summarized in Table S1.
A 20 mg/mL stock solution was diluted with PDA to obtain a series of concentrations, repeating the experiments above, and the inhibition rate was calculated respectively. The EC50 values were calculated by spss statistics v17.0 , and the results are illustrated in Table 1

Homology modeling and molecular docking
Homology modeling was carried out with the online program SWISS MODEL (http://swissmodel.expasy.org/). First, the primary amino acid sequence of CYP51 (Sclerotinia sclerotiorum organism) was uploaded to the program, then the SWISS-MODEL template library (SMTL version 2020-04-08, PDB release 2020-04-03) was searched with BLAST 3 and HHBlits 4 for evolutionary related structures matching the uploaded sequence (target sequence) when the Search For Templates was performed. Next, according to the reported result, the template was chosen for homology modelling of CYP51 crystal structure. Model was built based on the target-template alignment using ProMod3. Coordinates which are conserved between the target and the template are copied from the template to the model. Insertions and deletions are remodelled using a fragment library. Side chains are then rebuilt. Finally, the geometry of the resulting model is regularized by using a force field. In case loop modelling with ProMod3 fails, an alternative model is built with PROMOD-II . The global and perresidue model quality has been assessed using the QMEAN scoring function 5 . The built model was selected for the following molecular docking simulation. The templates for CYP51 was 6CR2 6 .
Molecular docking simulations of compounds (5a4, 5b2 and difenoconazole) against CYP51 were performed with Glide program (Schrö dinger, LLC: New York, NY, 2015) 7 . The protein structures were prepared using the Protein Preparation Wizard panel inserted in the Maestro with the default settings. Residues within 20 Å around the ligand were defined as binding sites in which the docking grids were generated by the Receptor Grid Generation panel. The default settings were adopted for the cutoff, neutralization, etc.. Finally, the prepared compounds were docked to the aforementioned docking girds with Standard precision (SP) mode. The top ranking pose of each compound was respectively selected for binding mode analysis. Figure S1. The alignment of target-template sequence A B Figure S2. A：The modeled structure of CYP51 of S. sclerotiorum in cartoon diagram.

B:
The modeled structure of CYP51 of S.sclerotiorum in cartoon diagram was superposed with the template structure.
The -helix, β sheet and loop were colored in cyan, magenta and salmon, respectively. The heme was shown as sticks representation.